Myosin, a major contractile protein in all cells, is composed of a dimer of two myosin heavy chains (MHCs) associated with two pairs of light chains. Smooth muscle myosin heavy chains exist as two isoforms (MHC204 and MHC200) that are generated from a single gene through alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P. and Periasamy, M.J., J. Biol. Chem. 264: 9734-9737, 1989). Our goal was to determine the isoform composition of the heavy chain in the native myosin molecule,to purify these isoforms and to characterize them biochemically. Antibodies specific for MHC204 were generated. These antibodies were used to analyze the association of the two MHC isoforms by immunoprecipitation from a solution of purified bovine aortic smooth muscle myosin which contained equal amounts of each. isoform. MHC204 quantitatively removed from this mixture was free of MHC200. Furthermore, a second round of immunoprecipitation with an antibody that recognizes both isoforms equally well, revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. The homodimeric organization of the heavy chain isoforms allowed us to purify myosin composed of only the 204 kDa heavy chain. Affinity purified antibodies to MHC204 were immobilized on a protein Gagarose HPLC column. Myosin composed of MHC204 homodimers was eluted using the synthetic peptide antigen employed to immunize the rabbits. The eluted MHC204 fraction was enzymatically active as judged by its ability to translocate actin in an in vitro motility assay. The velocity of movement of actin filaments by myosin consisting exclusively of 204 kDa heavy chains was significantly greater than that of myosin containing an equal mixture of MHC204 and MHC200. These results suggest heterogeneity in the enzymatic activity of these isoforms. Further examination of the biochemical properties of these isoforms promises to provide important clues for the reason for their existence.